The cytogenetic analysis of human cells and tissue material is typically based on microscopic inspection of banded metaphase chromosomes [Buckton and Evans 1973]. Cell samples taken from human tumors, however, usually contain too few cells in metaphase, so that mitogens have to be used to stimulate cellular proliferation. In solid tumors, stimulation of cellular growth of interphase cells is especially difficult or cannot be achieved at all [Gahrton et al. 1980; Trent 1985; Knuutila et al. 1986]. Cytogenetic analyses by means of in situ hybridization with chromosome-specific nucleic acid probes facilitate the differentiation between tumor cells and normal cells by allowing the analysis of interphase nuclei. Such analyses reduce the time and labor required for preparation of metaphase chromosomes and minimizes selection that may occur during cell culture [Cremer et al. 1986; Pinkel et al. 1986; Hopman et al. 1988; Trask et al. 1988; Nederlof et al. 1989].
Cloned probes suitable for chromosome-specific hybridization have now been reported for more than two-thirds of the human chromosomes. Some of such probes are specific for human satellite III DNA sequences [Cooke and Hindley 1979; Berdize 1987; Weier et al. 1990]. However, most of such probes bind to alpha satellite DNA found at or near the chromosome centromeres (pericentromeric) [Manuelidis 1978; Willard and Waye 1987]. The alphoid DNA sequences are comprised of tandemly repeated monomers of about 171 base pairs (bp) [Wu and Manuelidis 1980]. Certain parts of the 171 bp alphoid monomers appear to be conserved among all human chromosomes. Others, possibly organized as higher order repeats, show substantial chromosome-specific variation and may be used as the target of chromosome-specific probes [Devilee et al. 1986; Jorgensen et al., 1986; Murray and Martin 1987; Willard and Waye 1987]. Some authors have suggested that the chromosome-specificity is associated with organization of individual monomers in higher order repeats [Waye et al., 1987a and 1987b; Willard and Waye 1987; Hulsebos et al. 1988]. However, there is evidence that some monomers are sufficiently specific and so highly repeated that they can be used as a hybridization target for interphase chromosome enumeration [Meyne and Moyzis 1989].
Many of the probes reported so far allow ready analysis of chromosome copy number [Choo et al. 1990]. Others, however, show considerable cross-hybridization with non-target chromosomes and may require hybridization at elevated levels of stringency [Waye et al. 1987b; Devilee et al. 1988]. Under such conditions, signal intensities often decrease, so that such probes cannot be used in critical applications, for example, when hybridizing to highly condensed sperm chromatin [Wyrobek et al. 1990] or tissue sections wherein probe diffusion is poor [Emmerich et al. 1989] or when the hybridization target cannot be tightly controlled due partially to degradation of the cellular material.
Important probe parameters besides high specificity for the target chromosome type are the size of the probe molecule and the extent of the hybridization target area measured in base pairs (bp). For example, individual probe molecules may need to be of a size that favors diffusion into densely packed chromatin of sperm. Relatively short probe molecules that are complementary to highly reiterated DNA target sequences, such as the repeated satellite DNA, enable high signal intensities through binding of a large number of probe molecules to the target DNA without jeopardizing specificity. The preferred probes render highest possible signal-to-noise ratio.
The instant invention provides a primer directed DNA amplification method using the polymerase chain reaction (PCR) [Saiki et al. 1988b] with degenerate primers as an efficient means to isolate chromosome-specific repeated DNA. In the absence of any a priori knowledge other than the type of DNA repeat, for example, alpha satellite DNA, the methods of this invention allow the generation of chromosome-specific repeat sequence probe DNA. Disclosed are representative probes for human chromosome-specific alphoid DNA that have high specificity with high signal intensities in in situ hybridization experiments. In representative methods of this invention degenerate PCR primers for two conserved regions of the 171-bp alphoid monomer are used to amplify alphoid DNA from flow-sorted chromosome-specific DNA. The probes can be labeled by amplifying in the presence of modified dNTPs, or they can be labeled after completion of the PCR reaction by chemical or enzymatic modification of the PCR products.
Weier et al. (1990) described the use of in vitro DNA amplification for production of double-stranded, biotin-labeled DNA probes. In that article a 124 bp segment of the Y chromosome-specific 3.4 kb repeat was amplified from human genomic DNA using PCR with nondegenerate primers.
Koch et al. (1989) disclosed a DNA analysis method called Primed Amplification Labeling (PAL) in which biotin-labeled hybridization probes are produced in a polymerase chain reaction (PCR), in which two synthetic oligonucleotide primers anneal within the same alphoid monomer. If DNA from a specific chromosome is used as the template, Koch et al. reported that the resulting probe mixture "gives stronger and more chromosome-specific signals in in situ hybridization experiments than does a cloned alpha satellite DNA probe derived from the same chromosome." [Abstract, p. 259.]
The instant method differs from the Koch et al. PAL method in several substantial ways. The primers used by Koch et al. are different from those of the instant invention, not only in the location of the primer annealing sites within the consensus monomer and the direction of primer extension, but, more importantly, the Koch et al. primers are nondegenerate. Thus, the amplification scheme described by Koch et al. is likely to amplify a rather limited number of different alphoid DNA sequences, and under typical PCR conditions, would not allow amplification of the cloned DNA fragments, for example, those in pBS609-51 and pBS609-52 discussed infra which have base pair mismatches.
Specifically disclosed herein are representative probes for chromosome 8-specific and chromosome 10-specific alphoid DNAs. An alphoid DNA fragment, pJM128, that is enriched on human chromosomes 8 has been isolated from a human DNA library and is described in the literature [Donlon et al. 1987]. That probe, however, shows considerable crosshybridization with other human chromosomes complicating its use in interphase analysis [Donlon et al. 1987].
Further disclosed herein are experiments with unpurified representative probes demonstrating their specificity in competitive hybridization protocols with unlabeled genomic alphoid DNA and/or total genomic DNA. The use of such probes for enumeration of chromosomes in normal and tumor cell nuclei is also shown.
Probe DNA molecules prepared according to the methods of this invention were cloned and analyzed by a combination of in vitro DNA amplification, dideoxynucleotide sequencing and in situ hybridization. Probes were screened for specificity, repeat content and size, among other parameters. Representative monomeric probe molecules prepared according to the methods of this invention may have more than 80% homology with the alphoid consensus sequence or published alphoid monomers that map to different human chromosomes, but they show unprecedented specificity for highly reiterated DNA sequences on human chromosomes. The methods of this invention produce probes and collections of probes to highly repeated sequences such that the signal is much brighter and stronger than from a cloned repeat sequence probe that was prepared by conventional techniques.